B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties

B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form

B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating

Abstract

The present invention provides a stable stationary phase for chromatography which comprises porous ZrO₂ spherules coated with a polymer coating wherein said coated spherules have a pore size from about 20-500 Å and a particle diameter range of about 0.5-500 microns, and are stable in basic media to pHs of up to about l4.

Description

Currently-known inorganic chromatography supports comprising particulate silica (SiO₂) or alumina (Al₂O₃) are stable over pH ranges of about 1-8 and 3-12, respectively. The solubilization of SiO₂ and Al₂O₃ at pHs outside of these ranges results in deter­ioration of these supports and contamination of the resultant chromatographed and separated products with silicon- or aluminum-containing species. Methods of improving the alkaline stability of particulate SiO₂ by cladding the surface with a more base-stable metal oxide such as zirconium oxide (ZrO₂) have been dis­closed in U.S. Patent Nos. 4,648,975 and 4,600,646. This cladding is disclosed to increase the upper pH limit at which these supports, also referred to as packings, can be used to 11 and 9.5, respectively. However, these packings still lack adequate stability to allow them to be sterilized and cleaned in, for example, 0.1 N aqueous sodium hydroxide (NaOH, pH=13).

Use of porous spherical ZrO₂ particles on a thin layer chromatography plate has been disclosed in U.S. Patent No. 4,138,336, a process for the prepara­tion of porous ZrO₂ microspheres is taught in U.S. Patent No. 4,010,242, and chromatographic use of these particles is taught in U.S. Patent No. 3,782,075. The microspheres are prepared by a process in which colloidal metal oxide particles are mixed with a poly­merizable organic material and coacervated into spheri­cal particles by initiating polymerization of the organic material. This is a time consuming, batch pro­cess which requires the addition of organic material which is pyrolized and hence lost.

U.S. Patent No. 3,862,908 discloses micro­spheres of urania and other metal oxides, however, these particles are fired to near full density, have reduced surface areas and therefore, would not be attractive for chromatographic uses.

U.S. Patent No. 3,892,580 discloses a process for preparing porous bodies of ZrO₂. This process requires the use of a binder to react with the oxide particles during preparation. This binder is subse­quently decomposed by pyrolysis and therefore lost. The bodies produced by this process are not spherical, would pack unevenly, may cause increased column pressure, and are therefore not attractive for chroma­tographic uses.

U.S. Patent No. 4,389,385 teaches the prepara­tion of porous gels and ceramic materials by dispersing solid particles of an inorganic substance produced by a vapor phase condensation method in a liquid to form a sol. The sol contains colloidal particles which are aggregates of the primary particles. The sol is dried to produce a porous gel of greater than 70% by volume porosity.

B. Reverse Phase High Pressure Liquid Chromatography

The majority of separations employing high pressure liquid chromatography (HPLC) are performed in the so-called reversed-phase mode. In this mode, the column-packing material is referred to as stationary phase. The most commonly used stationary phases feature a non-polar ligand (e.g. octane or octadecane) covalently-bound to a porous silica particle through a siloxane bond (Si-O-Si) to render the surface hydro­phobic. Although these silica-based bonded phases are very useful for a wide range of applications in reversed-phase HPLC, their use is strictly limited to the pH range of between 2 and 8, due to the hydrolytic instability of both the silica support particle and the siloxane bond used to "anchor" the non-polar active group. Thus, the production of a pH-stable reversed-­phase support material must involve the development of both a stable, controlled porosity, high surface area support material and a method for rendering the surface permanently hydrophobic.

The eluent, also referred to as the mobile phase, used to elute the various components from the stationary phase is relatively polar, e.g., an aqueous buffer or a mixture of water and an organic solvent, e.g., aqueous alcohol. Its polarity can be changed by increasing the concentration of the less polar liquid in the mobile phase, a technique known in the art.

Thus relative to the use of ZrO₂-clad silica, a more promising approach to developing a highly stable reversed-phase support, involves replacing the silica with an alternative inorganic material, such as alumina. Although it has been demonstrated that some improvement in pH stability is realized by replacing silica with alumina, the dissolution of alumina in aqueous solutions at extreme pHs (pH<2 and pH>12), even at room temperature, is well known.

As mentioned previously, in addition to the use of a pH-stable support material, the production of a stable, reversed-phase also requires a process for modifying the support material which results in a stable, hydrophobic surface. Silylation is the most widely used method to derivatize silica particles to produce hydrophobic reversed-phase supports. The sily­lation of inorganic bodies other than silica (e.g. alumina, titania, zirconia, etc.) has been disclosed in U.S. Patent No. 3,956,179. However, it is uncertain whether or not covalent bonds to the support surface are actually formed. In any event, the hydrolytic in­stability of the siloxane bond is well known, and it is very likely that a Si-O-metal bond will be even more susceptible to aqueous hydrolysis because of the increased polarity of the bond.

An alternate approach to silylation for modi­fying the surface polarity of inorganic bodies is the sorption of a polymer of desired polarity/function­ality onto an SiO₂ or Al₂O₃ support surface followed by crosslinking of the individual polymer chains to one another to impart additional stability to the coating. Reversed-phase supports prepared in this fashion exhi­bit much improved pH stability compared to those pre­pared by silylation. It is important to recognize that the formation of a stable, crosslinked polymer layer on the surface of the support does not reduce the need for a stable, inorganic support, since it may not be possible to cover the entire inorganic surface. Although crosslinking of the polymer may keep it in place even as the underlying inorganic support dis­solves, dissolution of the support will undoubtedly lead to a reduction in the mechanical stability of the support. In addition, problems related to increasing column back pressure are known to accompany the disso­lution of the inorganic support and its subsequent appearance in the mobile phase and transport through the column and the accompanying instrumentation.

Another problem related to the use of silica-­based reversed phase supports is the difficulty encoun­tered in the chromatography of amines and other basic solutes. This problem results from the presence of acidic silanol groups (SiOH) on the silica surface. Basic solutes undergo very strong interactions with these silanol groups which may involve cation exchange or hydrogen bonding, depending on the pH of the mobile phase. Ths problem is exaggerated by the requirement of working in the pH range 2<pH<8 on silica-based columns, since most amines will be protonated in this pH range and protonated amines can readily bond to the silica surface. One obvious approach to improving the chromatography of amines is to work at hydrogen ion concentrations significantly above the ionization con­stant of the amines so that they are unprotonated. For aliphatic amines, this normally involves working at a pH greater than 11. However, these pH ranges cannot be employed using silica-based columns.

The presence of the aforementioned acidic silanol groups can also lead to irreversible adsorption of many classes of organic molecules onto silica-based reversed-phase supports, a problem which is well known to those versed in the art. This irreversible adsorp­tion is particularly troublesome in the reversed-phase HPLC of proteins. Ultimately, this adsorption will result in a change in the properties of the support and can lead to its destruction.

Reversed-phase HPLC is finding increased use in the area of bioprocessing because of HPLC's great ability to separate and purify materials. At the pre­parative scale, there are many unique considerations not applicable at the analytical scale. One such con­sideration is the need to sterilize a chromatography column prior to its use in the purification of a pro­duct intended for biological or human use.

Objects of the Invention

It is, therefore, an object of the present invention to produce chromatography column support material which is stable over a wide pH range, and which resists dissolution by aqueous media.

Furthermore, it is an object of the present invention to produce a support material comprising a non-polar surface which can be used for separation by both ion-exchange and reversed-phase processes, wherein the relative contribution of these two processes may be controlled by simple adjustment of mobile phase con­ditions.

Also, it is the object of the present inven­tion to produce a support material which can be regen­erated by freeing it from "irreversibly adsorbed" biological or organic residues by treatment at high pH.

It is another object of the present invention to provide a support material for use in large scale separations, particularly of products generated by biotechnology, for example, by fermentation, that can withstand traditional sterilization techniques involv­ing high pH and heat treatment.

Summary of the Invention

The present invention provides a support material adapted for use as the stationary phase in high-performance liquid chromatography (HPLC) which comprises porous spherules of zirconium oxide (ZrO₂, "zirconia"). These spherules display a remarkable phy­sical stability in aqueous media of a pH of about 1 to 14. Preferred ZrO₂ spherules are about 0.5-500 µ, most preferably about 1-20 µ in diameter, have a surface area of about 1-200 m²/g, most preferably about 40-150 m²/g; and have pore diameters of from about 0.5-500 Å, most preferably about 100-300 Å.

The ZrO₂ spherules of the invention are char­acterized in that they are prepared by a process con­sisting essentially of (a) dispersing an aqueous sol containing colloidal ZrO₂ particles in a liquid medium which extracts the water from the dispersed sol to afford gelled ZrO₂ spherules; (b) recovering the gelled spherules from the medium; and (c) heating the gelled spherules to yield solid porous ZrO₂ spherules. This process yields porous particles of ZrO₂ which are essentially spherical. When formed into a bed, the spherules provide improved mobile phase flow charac­teristics over those exhibited by irregularly-shaped, jagged-edged or angular particles.

In a preferred embodiment of this process, the colloidal ZrO₂ sol is centrifuged, the supernatant liquid decanted and the residue redispersed in an about equal volume of water. This procedure is preferably repeated a plurality of times (2-5X). The redispersed ZrO₂ yields spherules having a larger pore diameter and an increased pore volume, when they are formed in accord with the present method.

These particulate spherules can be formed into a bed, and employed as the stationary phase in separa­tions performed via chromatography. Therefore, the spherules can be used as the stationary phase in con­ventional chromatography columns which have an axial flow path, with or without rigid walls. For example, the ZrO₂ spherules can be packed into a column such as a HPLC column, where the packing functions as the stationary phase during HPLC separations which are accomplished by ion exchange and size exclusion pro­cesses. The spherules can also be used in columns which have a radial flow path or to form a fluidized bed, with single or multiple stage absorbers. The bed can also be formed of a mass of spherules which are contained in an immobilized enzyme reactor or other type of bioreactor.

The majority of HPLC methodologies involve use of the reverse phase mode, wherein the column-packing material (stationary phase) is non-polar, and the mobile phase is polar. Therefore, the present inven­tion also provides a support material comprising porous ZrO₂ spherules coated with a hydrophobic polymeric layer. The coated spherules are prepared by adsorbing a polymerizable monomer or oligomer onto the surface of the spherules and subsequently crosslinking it, e.g., by reaction of the adsorbed material with a free radi­cal initiator or by irradiation. The polymeric coating renders the ZrO₂ particles hydrophobic without substan­tially altering any of their desirable physical and mechanical properties. Likewise, the ZrO₂ spherules can be coated with a hydrophilic, crosslinked polymer to form an ion-exchange support material.

The coated spherules can also be combined with a suitable binder and used to coat a glass or plastic substrate to form plates for thin-layer chromatography.

Therefore, another preferred embodiment of the present invention is directed to a chromatographic support material comprising porous ZrO₂ spherules having a cross-linked polymeric coating thereon, wherein said coated spherules are hydrophobic, have a pore size from about 20-500 Å and an average diameter of about 0.5-500 µ.

As a result of the support material's remark­able stability over a wide pH range, it is useful for the chromatographic separation of compounds at their optimal pHs. For example, the coated material prepared in this fashion can be used for the separation of amines at a variety of pHs and mobile phase conditions such that the separation occurs either by a reversed-­phase retention mode, a cation-exchange mode, or some combination of the two. For example, at high pH (pH=12), the amines are unprotonated so that separation occurs entirely by a reversed-phase mode. At low pH in the presence of a low ionic strength phosphate buffer and with an organic solvent-rich mobile phase, the separation occurs via a cation-exchange mode. By adjustment of mobile-phase conditions, selectivity can thus be significantly adjusted.

The ZrO₂ spherules of the present invention can also be employed to immobilize bioactive materials for a variety of purposes, including catalysis, analy­sis, affinity chromatography and synthetic transforma­tions. Bioactive materials can be strongly sorbed onto the exterior and interior surfaces of both the uncoated and the polymer-coated ZrO₂ spherules, while retaining a large percentage of their initial bioactivity. Useful biomaterials include proteins such as enzymes and antibodies.

In addition, "irreversibly adsorbed" organic or biological residues can be removed from fouled columns packed with coated or uncoated spherules by flushing the column with a mobile phase at high pH or by injecting pulses of the high pH mobile phase. The term "irreversible adsorption" refers to the very strong tendency which surface-adsorbed proteins, bio-­polymers and the like exhibit to remain sorbed under normal elution conditions, until the mobile phase con­ditions are changed sufficiently to desorb them.

Therefore, coated or uncoated ZrO₂ spherules can be prepared which comprise a biologically active material such as an enzyme or a protein such as an immunoglobulin. Upon depletion of the biological acti­vity, the enzyme or other protein can be removed from the spherules by exposing them to an aqueous medium at high pH, e.g., by washing them with a solution of an alkali metal hydroxide. The spherules, stripped of the biological materials, can then be treated with a buffer to return them to a physiological pH, and subsequently reloaded with the same, or a different bioactive material.

The ZrO₂ spherules may also be exposed in situ to traditional sterilization conditions, for example, by exposing the packing or the packed column to heat and high pH, without significant degradation.

In a further preferred embodiment of the invention, the surface of the coated or uncoated ZrO₂ spherules is deactivated or modified by treatment with an effective amount of an organophosphonate prior to or following application of the hydrophobic polymer coating. It is believed that the organophosphonate becomes incorporated into the organic matrix of the polymeric coating.

Detailed Description of the InventionI. Zirconium Oxide

In the practice of this invention, a portion, or preferably a majority of the initial zirconium oxide (ZrO₂) used to form the present spherules is in the sol state; a colloidal dispersion of ZrO₂ particles. Such a state is clearly a different physical state than is achieved by the simple dispersion of a silica aerogel in water. The latter easily sediments or precipitates because of its relatively larger particle size and lack of stabilizing counter ions in solution. This is in contrast with a colloidal sol where the inorganic par­ticles in an aqueous solution usually are not visible to the naked eye.

Sol particles are submicron in particle size and hence will pass through most common filter papers. Sol particles in water do not aggregate because of a stabilizing electrical charge on the surface which is termed a zeta potential. Once the water is removed, the sol particles interact strongly with one another through hydrogen bonding and Van der Waals forces, to provide aggregated sol particles.

Colloidal dispersions of zirconium oxide suitable for use as the ZrO₂ source to prepare the pre­sent spherules are commercially available, e.g. as the Nyacol™ series, Nyacol, Inc., Ashland, MA. These dispersions contain about 20 wt-% ZrO₂, wherein the ZrO₂ particles vary in average diameter, e.g., from about 10-250 nm. For example, Nyacol™ Zr 95/20 is an aqueous dispersion containing 20 wt-% ZrO₂ of colloidal ZrO₂ particles, the majority of which are about 95 nm in diameter.

Non-colloidal ZrO₂ sources may be included along with the colloidal ZrO₂ particles used to prepare these spherules. Thus, chloride, nitrate, sulphate, acetate, formate or other inorganic or organic salts of zirconium such as the oxysalts and alkoxides may be included with the ZrO₂ sol and the mixture used to make spherules. In preferred mixtures, colloidal ZrO₂ par­ticles make up the bulk of the total ZrO₂ present.

Organic compounds may also be included with the ZrO₂ precursors used to prepare the spherules. These organic materials are fugitives which are removed during the firing of the spherules. In particular, water-soluble polymers such as polyvinylpyrolidone, polyethylene glycol, polyethylene oxide, and the like, or latex particles may be included in the liquid mix­ture used to prepare the spherules. These fugitives may be added to alter the rheology of the precursor solution or the pore structure of the resulting fired spherule.

It is also within the scope of the present invention to include precursors for other metal oxides with the ZrO₂ precursors so as to stabilize a particu­lar crystalline phase of ZrO₂ or to retard grain growth in the fired spherules. Thus, salts or sols of metals such as yttrium, magnesium, calcium, cerium, aluminum, and the like may be included in levels of from approxi­mately 0-20 mole-%. ZrO₂ spherules fired in air or in oxidizing atmospheres which do not contain other oxide additives display either monoclinic, tetragonal or pseudovibic crystal structures when cooled to room tem­perature. Higher firing temperatures and longer firing times favor the presence of the monoclinic phase. The inclusion of other metal oxides allows the preparation of spherules which possess either monoclinic, tetrago­nal, or cubic crystalline structures.

Those features of ZrO₂ are well known in the art and are discussed in, for example, An Introduction to Zirconia, Magnesium Elektron Ltd., Twickenham, England, (2d ed., Magnesium Elektron Publication No. 113, July 1986).

II. Preparation of ZrO₂ Spherules

To prepare the spherical ZrO₂, particles, or "spherules," of the present invention, an aqueous sol containing a colloidal dispersion of ZrO₂ particles is dispersed in a medium which can extract water from the dispersed sol in the form of droplets. Removal of all or a portion of the water results in gelled solid spherules which consist of aggregated sol particles. One medium which may be used is 2-ethyl-1-hexanol as disclosed in U.S. Patent No. 4,138,336. A preferred medium for safety reasons and ease of processing is peanut oil, which is preferably used at a temperature of about 80-100°C. The most preferred medium is a mix­ture of peanut oil and oleyl alcohol which are combined in a ratio of about 1:1, and used at a temperature of about 80-100°C. Oleyl alcohol possesses a higher extraction capacity than peanut oil and mixtures of the two allow the extraction capacity of the medium to be controlled. Depending upon the ratio of sol to forming medium extraction times of from about 1-60 minutes can be used to fully gel the ZrO₂ particles. The gelled spherules may be conveniently separated from the extracting medium, e.g., by filtration.

Once the ZrO₂ particles are condensed into spherules by the above process, thermal treatment at firing temperatures of from about 100-1500°C, prefer­ably about 400-800°C, is performed. The resulting fired spherules may be from about 1-500 µ is in diameter and can possess a surface area of 1-200 m²/g and pore diameters of from about 20-500 Å These particles have high mechanical strength and exceptional stability to aqueous solutions of pHs of about 1-14.

The particles may be packed into a HPLC column and used to perform HPLC chromatographic separations by ion exchange and size exclusion mechanisms. For a general discussion of HPLC techniques and apparatuses, see Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Publishing Co., Easton, PA (16th ed. 1980), at pages 575-576, the disclosure of which is incorporated by reference herein.

III. Polymer-Coated ZrO₂ Spherules

The majority of HPLC methodology employs the so-called "reverse phase" mode, i.e., the column-­packing material (stationary phase) is non-polar and the eluent (mobile phase) is polar. Therefore, it is preferred to coat the surface of the ZrO₂ spherules with a hydrophobic coating, which is also preferably stable to aqueous solutions having a pH of about 1-14. Hydrophilic polymer coatings can also be applied and crosslinked for modification of the ZrO₂ spherules to form an ion exchange support. These hydrophilic poly­mer coatings are formed from monomers or oligomers which comprise polar groups such as sulfonic acids, carboxylic acids, amino groups or quaternary ammonium groups. A preferred method to prepare such a coating comprises sorbing a polymerizable monomer or oligomer onto the surface of the spherules, and crosslinking the monomer or oligomer. See G. Shomberg, LC-GC, 6, 36 (1988).

A. Polymerizable Monomers or Oligomers

A wide variety of crosslinkable organic materials, which may be monomers, oligomers or poly­mers, can be employed to coat the porous ZrO₂ spher­ules. For example, such materials include polybutadi­ene, polystyrene, polyacrylates, polyvinylpyrrolidone (PVP), polyorganosiloxanes, polyethylene, polyethylene imine, polyaspartic acid and multifunctional silanes.

A preferred material for the preparation of a reversed phase support material is an oligomer of poly­butadiene. A preferred material for modification of the ZrO₂ spherules to form a cation ion exchange support is polyaspartic acid. A preferred material for construction of a support suitable for aqueous steric exclusion chromatography is a tri- or di-alkoxy-, gamma-glycidoxy silane.

B. Crosslinking Agents

Any of the common free radical sources includ­ing organic peroxides such as dicumyl peroxide, benzoyl peroxide or diazo compounds such as 2,2′-azobisisobuty­ronitrile (AIBN) may be employed as cross-linking agents in the practice of the present invention. Useful commercially available peroxyesters include the alkylesters of peroxycarboxylic acids, the alkylesters of monoperoxydicarboxylic acids, the dialkylesters of diperoxydicarboxylic acids, the alkylesters of mono­peroxycarbonic acids and the alkylene diesters of per­oxycarboxylic acids. These peroxyesters include t-­butyl peroctoate, t-butyl perbenzoate, t-butyl per­oxyneodecanoate and t-butyl peroxymaleic acid. These compounds are commercially available from Pennwalt Chemicals, Buffalo, NY. The amount of any free radical initiator required to catalyze the polymerization reac­tion will vary depending upon the molecular weight of the initiator and its thermal stability. Oligomers may also be polymerized by thermal treatment, by irradia­tion with UV light or gamma rays or by exposure to high energy electrons.

C. Coating/Crosslinking Process

Zirconium oxide may be modified in different ways to achieve materials with a light, intermediate or heavy carbon load. Preferably, the ZrO₂ spherules are first surface-hydrated and then dried in vacuo. Depending on the load desired, the dried ZrO₂ spherules are added to 15-50 ml of a pentane solution containing from 5-250 mg of an oligomer, such as polybutadiene, per gram of ZrO₂ spherules. The resultant slurry is placed in an ultrasonic bath and a vacuum applied in order to degas the particles and to insure that the oligomer solution has infiltrated substantially all of the pores. A free radical initiator, such as dicumyl peroxide, is then added at a level of 2-20% (w/w) rela­tive to the amount of polymer used. Solvent is then removed either by evaporation or by filtration, again depending on the desired carbon load. The treated ZrO₂ spherules are then heated to about 60-70°C under vacuum (10-20 mm Hg) for 12 hrs to remove any remaining sol­vent. The crosslinking reaction is then carried out by heating the coated ZrO₂ spherules in a tube furnace at 175-200°C for 2-4 hours under a blow of nitrogen.

Mixed-mode chromatography of amines can be performed in aqueous/organic mobile phases at various pHs containing different amounts of organic solvent, phosphate buffer and neutral salt for ionic strength adjustment.

A column "fouled" by repeated injections of large amounts of material, to the point that a marked change in characteristics is observed, can be stripped of irreversibly adsorped material. The original column performance can be restored by pulsing the column with 100 µl injection of 1 M NaOH or by flushing the column for about 0.5-10 hrs with aqueous alkali metal hydro­xide, i.e., with a 0.1 M NaOH solution.

The stability of the polymer-coated ZrO₂ spherules or uncoated ZrO₂ spherules to sterilizing conditions can be demonstrated by heating a previously characterized column to 100°C while pumping a 1 M NaOH solution through it for 12-24 hrs. Recharacterization of the column demonstrates that no significant change in column properties or decreased retention of a non-­polar substance has taken place.

IV. Bioactive Materials

A wide variety of bioactive materials can be bound to the uncoated or polymer-coated spherules by presently-available techniques so that their bioactiv­ity is retained and prolonged, or "stabilized" with respect to the unbound bioactive material. For example, antibodies or enzymes can be bound to the uncoated spherules in high concentrations by agitating an aqueous mixture of degassed spherules and antibody in a buffer, e.g., for about 0.1-5 hrs under ambient conditions. For a review of other noncovalent and covalent enzyme-binding methodologies, see R. A. Messing (U.S. Patent No. 3,850,751), the disclosure of which is incorporated by reference herein.

Enzymes capable of being bound and stabilized as described herein include a wide variety of enzymes which may be classified under six general groups: hydrolytic enzymes, redox enzymes, transferase enzymes, lyases, isomerases and ligases. The first group, hydrolase enzymes include proteolytic enzymes which hydrolyze proteins, e.g., papain, ficin, pepsin, tryp­sin, chymotrypsin, bromelin, keratinase; carbohydrases which hydrolyze carbohydrates, e.g., cellulase, glu­curonidase, amylase, maltase, pectinase, chitinase; esterases which hydrolyze esters; e.g., lipase, choli­nesterase, lecithinase, phosphatase; nucleases which hydrolyze nucleic acid, e.g., ribonuclease, deoxyribo­nuclease; and amidases which hydrolyze amines, e.g. arginase, aspariginase, glutaminase, and urease. The second group are redox enzymes that catalyze oxidation or reduction reactions. These include glucose oxidase, catalase, peroxidase, lipoxidase, and cytochromes. The third group are transferase enzymes that transfer groups from one molecule to another. Examples of these are glutamic-pyruvic transminase, glutamic-oxalacetic transaminase, transmethylase, phosphopyruvic trans­phosphorylase and dehydrogenase. The fourth group are lyase enzymes that catalyze the cleavage of C-C, C-O, C-N and other bonds by elimination, leaving double bonds, or conversely, adding groups to double bonds. Examples of these are pyruvate decarboxylase, amino acid decarboxylases, aldolase, fumarate hydratases, aconitate hydratases and ammonia lyase. The fifth group are isomerase enzymes that catalyze the dehydro­genation and epimerization of amino acids and sugars. An example of an isomerase is phosphoglucomutase. The sixth group are ligase enzymes that catalyze the syn­thetic linking of two molecules, simultaneously with the breakdown of ATP. Examples of these are aminoacyl-tRNA synthetases and biotinyl-dependent car­boxylases.

Other proteins capable of being bound and stabilized as described herein include Con-A, Protein-A, plasma immunoglobulins, monoclonal antibod­ies, bioactive polypeptides such as serum proteins and immunomodulators, e.g., lymphokines and the like. Other examples of proteins which are bound by the pre­sent spherules as provided in the working example, hereinbelow.

V. Phosphate Modification

The surface of uncoated or polymer-coated ZrO₂ spherules can be easily and dramatically modified in a chromatographically-beneficial way by treatment with aqueous inorganic phosphate solutions. The interaction between polymer-coated ZrO₂ and phosphate produces a mixed mode stationary phase exhibiting both cation-­exchange and reversed-phase properties. This allows one to adjust the selectivity of the present support material with respect to a group of basic solutes by appropriate adjustment of mobile phase pH, ionic strength, and reversed-phase eluting strength (i.e., volume fraction of the adjuvant organic solvent).

For some applications, it is desirable to further deactivate or modify the surface of the uncoated or polymer-coated ZrO₂ spherules. This can be used to enhance the ability of the spherules to separate polar compounds such as amines and carboxylic acids, or to modify retention. This deactivation can be accomplished by treating the uncoated ZrO₂ spherules with an organophosphorus compound in a suitable solvent for the organophosphorus compound. Useful organophos­phorus compounds include allylphosphonate, octyl phosphonate, phenyl phosphonic acid, napthyl phosphonic acid, phenyl phosphinic acid, phenylphosphoric acid and diallyl phosphorate. Preferred organophosphorus com­pounds include the unsaturated organophosphonic acids and the water-soluble salts thereof.

Useful solvents for the organophosphorus com­pound include aqueous alcohol, e.g., a solution of water and a (C₁-C₅)alkanol. The ZrO₂ spherules are preferably coated by agitating the spherules in a solu­tion of the organophosphorus compound so that the weight ratio of the organophosphorus compound to spherules is about 0.25-1:1. The treated particles are then separated from the treating solution, dried and the crosslinked polymeric coating applied as discussed hereinabove.

The invention will be further described by reference to the following detailed examples.

Example 1.

Peanut oil (3 liters) was placed in a 4 liter beaker and heated to 90°C. A mechanical agitator was inserted and the peanut oil was vigorously stirred. One hundred grams of Nyacol™ Zr 95/20, a colloidal ZrO₂ manufactured by Nyacol, Inc. and containing 20 wt-% of ZrO₂, primarily as about 95 nm particles, was sprayed into the peanut oil through an aerosol atomizer. After approximately 30 minutes, the batch was filtered through a No. 54 Whatman filter. Approxi­mately 17 g of solids were recovered, which were predo­minately spherules having a diameter of < 30 µ.

Example 2.

Peanut oil (600 g) and 600 g of oleyl alcohol were mixed and heated to about 90°C. Under vigorous agitation, 100 g of Nyacol™ Zr 95/20 was sprayed into the peanut oil/oleyl alcohol mixture as described in Example 1. After 30 minutes, the batch was filtered and the particles collected. The particles were predo­minantly (ca: 70%) spherules having a diameter of < 50 µ.

Spherules prepared as described in Examples 1 and 2 were thermally treated at a series of tempera­tures and the surface area, average pore diameter and pore volume were measured by nitrogen adsorption isotherm on a Quantasorb surface area analyzer. These results are summarized in Table I, below.
Table I

Firing Temp (°C)*

Surface Area (m²/g)

Average Pore Diameter {Å)

Pore Volume (%)

400

142

42

47

500

92

71

50

600

34

110

36

800

17

205

34

900

14

220

31

* 6 hrs

The data summarized on Table I show that it is possible to increase the average pore diameter by increasing the firing temperature from 400 to 900°C. The surface area and pore volume decrease with increas­ing firing temperature. Chromatographic activity of the ZrO₂ spherules is determined by the parameters of surface area, average pore diameter and pore volume. Accordingly, the appropriate firing temperature is selected.

Example 3.

The procedure of Example 2 was used to prepare spherules using Nyacol™ Zr 50/20, a colloidal ZrO₂ supplied by Nyacol, Inc. (50 nm ZrO₂ colloid size) as the ZrO₂ source.

Example 4.

The procedure of Example 2 was used to prepare spherules using Nyacol™ Zr 150/20, a colloidal ZrO₂ supplied by Nyacol, Inc. (150 nm ZrO₂ colloid size) as the ZrO₂ source.

Table II summarizes the surface area, average pore diameter and pore volume of spherules prepared as per Examples 2-4 and fired at 600°C for 6 hrs.
Table II

ZrO₂ Source*

ZrO₂ Colloid size (nm)

Surface Area (m²/g)

Average Pore Diameter (Å)

Pore Volume (%)

Zr 50/20

50

33

92

31

Zr 95/20

95

34

110

36

Zr 150/20

150

40

147

45

*Nyacol™ series.

The data summarized in Table II show that it is possible to control the average pore diameter of the fired spherules by appropriate selection of the colloid size of the ZrO₂ source. Larger colloids produce fired spherules with larger pore diameters and pore volumes.

Example 5.

Nyacol™ Zr 95/20 colloidal ZrO₂ was placed in a laboratory centrifuge and sedimented. The superna­tant was decanted and discarded. The sedimented ZrO₂ was redispersed in an equal volume of distilled water. Spherules were prepared from this centrifuged sol following the procedures of Example 2.

Example 6.

The centrifugation procedure of Example 5 was performed and the redispersed sol was subsequently recentrifuged to sediment, the supernatant decanted off and the ZrO₂ redispersed. Spherules were prepared from this doubly centrifuged sol following the proce­dure of Example 2.

Example 7.

The double centrifugation procedure used in Example 6 was performed and the redispersed sol was subsequently recentrifuged to sediment, the supernatant decanted, and the ZrO₂ redispersed. Spherules were prepared from this triply centrifuged sol following Example 2.

Centrifugation, removal of the supernatant, and redispersion of the colloidal ZrO₂ starting material results in increases in the average pore diameter, pore volume and surface area of fired spherules. This increase is believed to result from the removal of small (ca. 5-10 nm) colloidal ZrO₂ par­ticles which are known to be present in the Nyacol™ Zr series sols as a minor component. Many of these smaller ZrO₂ particles remain suspended during centri­fugation and are removed when the supernatant is dis­carded prior to redispersion of the larger sedimented ZrO₂ particles. If present, these small ZrO₂ par­ticles are believed to increase the packing density of the spherules by filling the interstices between larger ZrO₂ particles and therefore decreasing the average pore diameter, pore volume and surface area of the fired spherules.

It is also possible that sedimentation by centrifugation may result in agglomeration of the colloidal ZrO₂ particles into aggregates which pack together in a more open structure (effectively behaving as larger particles) than unaggregated particles.

Regardless of mechanism, the centrifugation treatments described in Examples 5-7 provide a method of preparing spherules with increased average pore dia­meter, pore volume and surface area relative to spherules prepared from untreated colloidal ZrO₂ sols.

The following example demonstrates the use of the unmodified ZrO₂ spherules prepared as described above in the chromatographic separation of proteins.

Example 8.

ZrO₂ spherules prepared as described in Example 2 were heated to 600°C for 6 hrs. The spherules were classified and the 5-10 µ fraction was used. The surface area of the spherules was 55 m²/g and the average pore diameter was 146 Å. The ZrO₂ spherules were slurried in methanol and packed into a 0.46 x 30 cm stainless steel column at a constant pressure of 4,000 psi to rapidly compress the ZrO₂/­methanol slurry to yield a uniform packing. After packing, the flow was maintained at 1 ml/min. at 1,000 psi. The column was washed with 150 ml of 100 mM sodium phosphate, pH 7.0. All subsequent chromato­graphy was performed in phosphate buffer. The column was stored in 20% methanol/water.

Protein solutions (2 mg/ml) were prepared in the same phosphate buffer: 20 µl samples of bacitracin, 1.4 KDa; ovalbumin, 45 KDa; and bovine serum albumin (67 KDa) samples were injected and eluted with 30 ml of buffer. All chromatographic runs were performed with a Spectra Physics Model 8700XR HPLC system with their Model 757 variable wavelength detector set at 280 nm. Elution profiles, peak areas and elution volumes were recorded on a Model 4290 integrator/recorder. The pro­teins eluted as shown in Table IV, below, consistent with results expected in exclusion chromatography.
Table IV

Protein

Elution Volume (ml)

Bacitracin

3.35

Ovalbumin

2.51

Bovine Serum Albumin

2.38

Example 9.

ZrO₂ spherules prepared as described in Example 2 were heated to 600°C for 6 hrs. Particles in the 30-40 µ diameter range having a surface area of 30 m²/g and an average pore diameter of 100 Å were used. The spherules were hand-packed into a 5 cm x 0.21 cm column via a methanol slurry. After packing, the column was washed for 12 hrs at 0.2 ml/min with pH 7.0, 50 mM phosphate buffer. All subsequent chromatography was done on an IBM 9533 L.C. at a flow rate of 1 ml/min and used a pH gradient of 50 mM H₃PO₄ at pH 2.0 to 50 mM Na₂HPO₄ at pH 10 over a time of 10 min followed by an additional 10 min of isocratic operation at pH 10 with 50 mM Na₂MPO₄. Bovine serum albumin (BSA) and myoglobin were separated by adsorption and ion exchange chromatography, yielding retention times of 13.3 min (BSA) and 17.8 min (myoglobin).

The anion exchange capacity for adsorption of picric acid was determined to be 230 µmoles/g of modi­fied ZrO₂. This substrate was used to separate oval­bumin from BSA. The column was operated with a gradient of 10 mM Tris buffer at pH 7.5 to 10 mM Tris at pH 7.5 with 0.5 M NaCl over 20 min, followed by an additional 10 min of isocratic operation at pH 7.5 with 0.5 M NaCl. The flow rate was 1 ml/min. The retention times were 9.75 (ovalbumin) and 22.8 min (BSA).

The following examples demonstrate the use of the ZrO₂ spherules to immobilize proteins.

Example 11.

ZrO₂ spherules with a diameter of approxi­mately 30 µ is and a surface area of 50 m²/g and an average pore diameter of 124 Å were used. Mouse anti­human IgE antibody was purified and radioiodinated (I¹²⁵) by the method of S. M. Burchiel et al., J. Immunol. Meth., 69, 33 (1984); K. L. Holmes et al., PNAS USA, 82, 7706 (1985), and diluted with unlabelled antibody to yield a specific radioactivity of 5,000 cpm/µg. A portion of 250 µl of antibody (250 µg/ml in 5 mM Tris, pH 8.0) was added to tubes containing 10 mg of spherules. The mixture was briefly evacuated, then rocked at ambient temperature for the appropriate time, 5-120 min., with three replicates for each time point. The tubes were centrifuged and rinsed twice with 1 ml of buffer. The spherules were transferred to a fresh tube along with 2 ml of buffer, the buffer removed and the radioactivity of each tube was determined in a Packard Model 5230 gamma scintillation counter. The amount of bound protein in ng, converted from cpm, is shown in Table V.
Table V

Time (Min)

Antibody Bound/mg Spherules

10

54 ng

20

66 ng

30

72 ng

60

69 ng

120

62 ng

Example 12.

Using the same materials and techniques des­cribed in Example 11, the extent of binding of antibody in 2 hr incubations as a function of its concentration (1-250 µg/ml) was determined. The averages of three replicates show a saturation (Table VI). Double-­reciprocal analysis of these data extrapolate to 100 µg antibody bound per g spherule at saturation.
Table VI

Conc. Protein (µg/ml)

Antibody Bound/mg Spherules

1

1.5 ng

5

7.5 ng

10

14.0 ng

50

38.0 ng

250

62.0 ng

Example 13.A. Trypsin Immobilization.

Solutions (2 mg/ml) of trypsin, a 24 KDa pro­teolytic enzyme and bovine serum albumin (BSA), a 67 KDa protein, were bound to 70 mg of the ZrO₂ spherules (average pore diameter 100 Å surface area of 30 m²/g) in 5 mM tris, pH 8.0 by agitating the degassed spherules in 1.0 ml of buffer for 17.5 hr. Trypsin (15.3 mg) and <0.2 mg (BSA) bound per g of spherule, a proportion which might be expected from their relative sizes and the size of the pores.

Trypsin was assayed using the thioesterase assay disclosed by P. L. Coleman et al., Meth. Enzymol., 80, 408 (1981). The bound spherules were suspended in 1 ml of buffer and a 5 µl aliquot was added to a tube containing 1.0 ml of substrate. After 2.5 min of continuous shaking, a citrate-soybean tryp­sin inhibitor (STI) solution was added to quench the reaction. It was rapidly centrifuged and the super­natant removed for determination of the absorbance (A) at 412 nm. Assays were performed on trypsin spherules, BSA spherules and the supernatant from trypsin spheru­les. The assay was also performed with the trypsin inhibitor in the substrate solution to determine whether it was able to inhibit the bound trypsin. The results of these assays are summarized on Table VII, below.
Table VII

Sample

Trypsin Activity (A at 412 nm)

-STI

+STI

Trypsin spherules

2.36

1.79

BSA spherules

0.10

0.13

Trypsin supernatant

0.19

0.12

The results (Table VII) indicate that about 75% of the bound activity is unavailable to STI, even though STI is smaller than trypsin. In addition, only 4% of the activity is attributable to unbound trypsin, a surprisingly low value given the inefficient batch washing method which was used.

Calculations based on these observations demonstrated several unexpected results. For example, 15 mg of trypsin/g ZrO₂ corresponds to 51 mg/ml using 3.3 g/ml as the density of the spherules. This corres­ponds to a trypsin concentration of 2 mM in the column. A check on this may be made by estimating the expected A at 412 nm for the assay. In these assays, the spherule-bound enzyme was 0.21 µm, the kcat for the substrate is 75/sec [G. D. J. Green et al., Anal. Biochem., 93, 223 (1979)] and the extinction coeffici­ent is 14,100, yielding an estimated 3.3 absorbance change, which compares favorably with the 2.4 observed. Since chromogen was present in amount sufficient to give only 2.8 A at 412 nm, it is safe to assume that nearly all of the bound trypsin is active. Thus, an extraordinary amount of protein is bound and retains its enzymic activity.

B. Chymotrypsinogen-Chymopapain-BSA Immobil­ization.

The procedure of Example 7 was employed to prepare ZrO₂ spherules having 240 Å pores and a surface area of 27 m²/g. Small columns were poured, each con­taining about 1.0 g of spherules, and were equilibrated with either 20 mM tris-chloride buffer (pH 8.0) or 50 mM sodium acetate buffer (pH 4.5). Chymotrypsinogen (24.5 kDa) and chymopapain (32 kDa) were dissolved in the tris buffer and BSA was dissolved in the acetate buffer. Protein-containing solution was continuously added to the column until the 280 nm absorbance of the eluate equalled that of the starting solution. Unbound protein was rinsed from the column, and the amount of bound protein was calculated from the difference between that added and that recovered in the eluate.

Chymotrypsinogen and chymopapain bound at 76.9 mg and 24.5 mg of protein/g of spherules at pH 8.0, respectively, and 64 mg of BSA bound per gram of spherules at pH 7.5. Converting these values into binding densities per ml of column volume yields 254, 81 and 211 mg/ml of protein, respectively.

The fact that at acidic pH, albumin binds to a greater extent than does the smaller chymopapain, and almost to the extent as the even smaller chymotrypsino­gen suggests that the latter enzymes would bind to even greater densities at lower pH, i.e., below their pIa.

A solu­tion of 0.55 g of polybutadiene (Aldrich Chemical Co., Milwaukee, WI, m.w. 4500, Cat. No. 20-050-6) in 50 ml of pentane was added to 3.5 g of ZrO₂ spherules pre­pared as described in Example 2 (fired at 600°C for 6 hrs; particle size = 20-45 microns) which had been boiled in CO₂-free water to fully hydrate the surface and then dried at 125°C. The slurry was placed in an ultrasonic bath and a water aspirator vacuum applied. Dicumyl peroxide (DCP) (0.01 g) was then added and the slurry was again placed in an ultrasonic bath and a vacuum applied. The pentane was removed in vacuo and the material dried at 70°C under vacuum. The material was then heated in a tube furnace at 200°C for 2 hrs and then washed successively with pentane, toluene, methylene chloride, tetrahydrofuran, methanol and 0.1 M sodium hydroxide. Elemental analysis of the coated spherules showed a carbon load of 7.7%. A duplicate sample was prepared in an identical fashion and had a carbon load of 7.5%. Because of the extremely heavy load of polybutadiene, the specific surface area of the porous spherules, as determined by a BET measurement, decreased from 50.4 to 4 m²/gm.

Preparation B - Lightly loaded ZrO₂:

35 ml of a solution of 0.09 g of polybutadiene in pentane was added to 3.5 g of ZrO₂ spherules and the resultant slurry was placed in an ultrasonic bath and a water aspirator vacuum applied. Pentane (10 ml) containing 0.002 g of DCP was then added and the slurry was again placed in an ultrasonic bath and a vacuum applied. The slurry was then shaken for one hr and the supernatant removed by filtration. The material was then washed as described in Preparation A. Elemental analysis of the coated spherules showed 0.84% carbon, while the BET results showed a specific surface area of 38.7 m²/gm. The decrease in specific surface area from 50.4 to 38.7 m²/gm is similar to the reduction in surface area which occurs upon silylation of typical inorganic supports.

Preparation C - Intermediate load:

A solution of 0.27 g of PBD in 50 ml pentane was added to 3.0 g of ZrO₂ spherules (mean particle diameter 3.5 microns). The slurry was placed in an ultrasonic bath and a vacuum applied. 5.2 mg of DCP in 10 ml of pentane were then added. The methodology of Preparation A was then followed. Elemental analysis showed 2.7% carbon.

It is clear from the results of carbon analy­sis that carbon had been deposited on the surface of the ZrO₂ spherules. Figure 1 further demonstrates the reversed-phase nature of the polymer-modified ZrO₂ spherules as exhibited by a 5 cm x 0.46 cm column packed using Preparation C. The linearity of the log k′ (capacity factor) vs. carbon number plot for the members of a homologous series of alkylphenones is clearly indicative of a reversed-phase retention mechanism.

Example 15.

A mixed cation-exchange/reversed phase support was prepared by treating a material prepared as des­cribed in Example 14, Preparation C with a 100 mM aqueous H₃PO₄ solution at pH 3 for about one hour. The retention data given in Table VIII show distinct changes in selectivity as a function of pH, volume fraction organic solvent and mobile phase ionic strength.
Table VIII

Separations at high pH (above the pKa of the amines) are dominated by a reversed-phase retention mechanism as are separations at lower pH in high ionic strength mobile phase. Conversely, separations at low pH in a low ionic strength environment are controlled primarily by cation-exchange processes. In addition to the ability to alter selectivity in several ways, the sub­ject material also exhibits dramatic improvement in terms of the peak symmetry of amine solutes relative to silica.

Example 16.

The pH stability of the material of Example 14, preparation A, has been demonstrated in chromatographic experiments at high pH and elevated temperature by monitoring the retention of test solutes and by measurement of the amount of carbon on the support before and after prolonged exposure to high pH. These experiments were carried out under the following chromatographic conditions: Mobile Phase A: 0.1 M CO₂-free NaOM; Mobile Phase B: Methanol; Flow Rate: 1 ml/min; Oven Temp: 50°C.

The retention of two test solutes in a mobile phase of 50% B/50% A as a function of the number of column volumes of mobile phase flushed through the column is shown in Figure 2A. Note that the initial decrease in retention reflects the equilibration of the column to the elevated temperature and not a loss in bonded phase. The evaluation was repeated on the lightly loaded material (Preparation B); the retention data on this material is shown in Figure 2B. Once again, there is an initial decrease in retention asso­ciated with column equilibration. There is also a slight decrease in retention at approximately 15 hours which accompanied a change in the lot of mobile phase; this change does not reflect a significant drop in car­bon load.

It is believed that the above evaluations represent the most challenging test of pH stability which has been reported for any reversed-phase material and it is also believed that the data clearly show that the spherules of Example l4, Preparations A and B, are essentially stable under these conditions.

Example 17.

ZrO₂ spherules prepared by the procedure of Example 2 (3.4 g, surface area: 60 m²/g; pore diameter: 95 Å) were treated with a solution of 1.6 g of allyl­phosphonate in 50 ml of 95/5 (v/v) methanol/water. After "ultrasonicating" under vacuum and shaking for one hr, the supernatant was removed by filtration and the phosphonate-treated ZrO₂ was dried at 70°C for 12 hrs. The material was then modified with PBD according to Example 14, Preparation C. In this manner, the residual ZrO₂ surface was deactivated as is clearly shown by the data in Table IX. Note that carboxylic acids are not eluted on the non-phosphonated ZrO₂ material but are eluted on the phosphonated material.
Table IX

Solute

k′(untreated)

k′(treated)

Toluene

0.46

0.49

Benzoic Acid

not eluted

6.1

Example 18.

Several 100 µl injections of cytochrome C were made on a column packed with material prepared as des­cribed in Example 14, Preparation C. The retention of cytochrome C on this material decreased due to "irreversible" adsorption of protein upon each injec­tion.

The column was then "pulsed" with 5, 100 µl injections of 1 M NaOM in order to strip the "irrever­sibly adsorbed" cytochrome C. The effect of the pulses is to strip the column of adsorbed protein such that the original retention characteristics can be regener­ated.

Example 19.

The ability of the subject matter to withstand sterilizing conditions was demonstrated by taking a material prepared as described in Example 14, Prepara­tion C, and evaluating its chromatographic characteris­tics, by exposing it to 1 M NaOM at 100°C for 1 hr and then reevaluating the chromatographic properties. As shown by the data in Table X, there was no decrease in retention of nonpolar substances upon challenging the packing in this fashion.
Table X

k′

Solute

Before Treatment

After Treatment

Benzene

1.36

1.47

Toluene

2.68

3.01

Ethyl Benzene

4.83

5.57

Propyl Benzene

9.21

10.86

A second column (ES Industries, Marlton, NJ) which uses an alumina support modified by the method of G. Shomberg, LC-GC, 6, 36 (1988), was challenged with a mobile phase of 1M NaOM, which was collected in two fractions. The first corresponded to an elution time of 1 hr and the second to an additional elution of 2.25 hrs.

The elements were analyzed via an inductively coupled plasma spectrometer. The concentration of aluminum- in the eluent from the second column corres­ponded to the dissolution of a total of 10% of the mass of the alumina in the column.

In marked contrast, zirconium was absent at a level of detectability of 0.03 µg/ml. Even if Zr was present at the detection limit, this would correspond to loss of less than 0.001% of the mass of ZrO₂ on the test column.

The invention has been described with refer­ence to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remain­ing within the spirit and scope of the invention.

Claims (18)

1. A bed comprising porous ZrO₂ spherules of about 20-500 microns in diameter, about 1-200 m²/g sur­face area and of about 0.5-500 Å pore diameter; wherein said spherules form the packing of an HPLC column and are substantially stable in an aqueous solution of a pH of about 1-14.

2. The bed of claim 1 wherein the spherules are about 45-500 microns in diameter and have a surface area of about 40-150 m²/g and an average pore diameter of about 100-300Å.

3. The bed of claim 1 wherein the porous ZrO₂ spherules are characterized by having been prepared by a process consisting essentially of:

(a) centrifuging an aqueous sol containing colloidal ZrO₂ particles to yield a residue of ZrO₂ particles and a supernatant;

(b) recovering said residue;

(c) redispersing said residue in an about equal volume of water to form an aqueous sol con­taining colloidal ZrO₂ particles;

(d) dispersing the aqueous sol in a liquid medium which extracts the water from the sol to afford gelled ZrO₂ spherules;

5. Porous ZrO₂ spherules coated with a hydrophobic crosslinked polymer coating wherein said coated spherules have a pore size from about 20-500 Å and a particle diameter range of about 0.5-500 microns, and are stable to about pH 14 in aqueous media.

7. The spherules of claim 5 wherein the surface of the ZrO₂ spherules is modified by treatment with inorganic phosphate to impart both cation-exchange and hydrophobic characteristics to said spherules.

8. The spherules of claim 7 wherein the inorganic phosphate is H₃PO₄.

9. The spherules of claim 5 wherein the surface of the ZrO₂ spherules is deactivated by treatment with an effective amount of an organophosphorus compound prior to application of the hydrophobic polymer coating.

10. The ZrO₂ spherules of claim 9 wherein the surface of the coated spherules is deactivated by treatment with an effective amount of an unsaturated organo­phosphonate or organophosphonic acid.

11. The ZrO₂ spherules of claim 10 wherein the organo­phosphonate is allylphosphonate.

12. The ZrO₂ spherules of claim 5 wherein the surface of the coated spherules is deactivated by treatment with an effective amount of an organophosphorus compound following application of the hydrophobic polymer coating.

13. The ZrO₂ spherules of claim 5 wherein the surface of the coated spherules is deactivated by treatment with an effective amount of an unsaturated organo­phosphonate or organophosphonic acid.

14. The ZrO₂ spherules of claim 13 wherein the organo­phosphonate is allyl phosphonate.

15. A thin layer chromatography plate comprising a substrate coated with a mixture of a binder and a particulate support material comprising the spheru­les of claims 5, 7, 10 or 12.

16. The bed of claim 1 wherein the surface of the ZrO₂ spherules is modified by treatment with inorganic phosphate or with an organophosphorus compound.

17. A bed comprising porous ZrO₂ spherules of about 20-500 microns in diameter, about 1-200 m²/g sur­face area and of about 0.5-500 Å pore diameter; wherein said spherules are substantially stable in an aqueous solution of a pH of about 1-14, and comprise an immobilized protein.

18. The bed of claim 17 which comprises an enzyme immo­bilized on said spherules.